Vascular effects of sildenafil in patientswith pulmonary fibrosis and pulmonaryhypertension: an ex vivo/in vitro study

Javier Milara1,2,3,4,8, Juan Escrivá5,8, José Luis Ortiz1, Gustavo Juan4,6,Enrique Artigues7, Esteban Morcillo1,4 and Julio Cortijo1,2,3,4

Affiliations: 1Dept of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain. 2ClinicalResearch Unit (UIC), University General Hospital Consortium, Valencia, Spain. 3Research Foundation ofUniversity General Hospital of Valencia, Valencia, Spain. 4CIBERES, Health Institute Carlos III, Valencia, Spain.5Thoracic Surgery Unit, University and Polytechnic Hospital La Fe, Valencia, Spain. 6Respiratory Unit,University General Hospital Consortium, Valencia, Spain. 7Surgery Unit, University General HospitalConsortium, Valencia, Spain. 8Both authors contributed equally to this work.

Correspondence: Javier Milara, Unidad de Investigación, Consorcio Hospital General Universitario, Avenidatres cruces s/n, E-46014 Valencia, Spain. E-mail: xmilara@hotmail.com

ABSTRACT Sildenafil improves the 6-min walking distance in patients with idiopathic pulmonaryfibrosis (IPF) and right-sided ventricular systolic dysfunction.

We analysed the previously unexplored role of sildenafil on vasoconstriction and remodelling ofpulmonary arteries from patients with IPF and pulmonary hypertension (PH) ex vivo. Pulmonary arteriesfrom 18 donors without lung disease, nine IPF, eight PH+IPF and four PH patients were isolated tomeasure vasodilator and anti-contractile effects of sildenafil in isometric organ bath. Ventilation/perfusionwas explored in an animal model of bleomycin lung fibrosis.

Sildenafil relaxed serotonin (5-HT) pre-contracted pulmonary arteries in healthy donors and IPFpatients and, to a lesser extent, in PH+IPF and PH. Sildenafil inhibited 5-HT dose-response contractioncurve mainly in PH+IPF and PH, but not in healthy donors. Sildenafil did not impair the ventilation/perfusion mismatching induced by bleomycin. Pulmonary arteries from PH+IPF patients showed amarked expression of phosphodiesterse-5 and extracellular matrix components. Sildenafil inhibitedpulmonary artery endothelial and smooth muscle cell to mesenchymal transition by inhibition ofextracellular regulated kinases 1 and 2 (ERK1/2) and SMAD3 phosphorylation.

These results suggest an absence of direct relaxant effect and a prominent anti-contractile and anti-remodelling role of sildenafil in PH+IPF pulmonary arteries that could explain the beneficial effects ofsildenafil in IPF with PH phenotype.

@ERSpublicationsSildenafil prevents pulmonary vasoconstriction and remodelling in patients with pulmonaryhypertension and fibrosis http://ow.ly/XU3ig

Copyright ©ERS 2016

Editorial comment in Eur Respir J 2016; 47: 1615–1617.

This article has supplementary material available from erj.ersjournals.com

Received: July 29 2015 | Accepted after revision: Jan 23 2016 | First published online: March 23 2016

Support statement: This study was funded by CIBERES (CB06/06/0027), Fondo de Investigaciones Sanitarias (PI14/01733), the Regional Government of Valencia (Prometeo II/2013/014), Pfizer and the Spanish Government (ADE10/00020, SAF2012-31042 and SAF2014-55322-P). Funding information for this article has been deposited with FundRef.

Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com

Eur Respir J 2016; 47: 1737–1749 | DOI: 10.1183/13993003.01259-2015 1737

ORIGINAL ARTICLEPULMONARY VASCULAR DISEASES

IntroductionIdiopathic pulmonary fibrosis (IPF) is a fatal disorder with a median survival time of 2.5–5 years afterdiagnosis. Pulmonary hypertension (PH) is recognised as a severe complication of IPF associated with poorsurvival [1]. Recent epidemiological studies reported a 32–84% prevalence of PH in patients with IPF,depending on the stage of IPF progression [1]. PH as a complication of IPF is associated with reduced exerciseability, as evidenced by decreased 6-min walking distance (6MWD) and increased oxygen desaturation.

The pathogenesis of PH in patients with IPF is characterised by vascular muscularisation in the earlystages, followed by fibrous vascular atrophy and pronounced intimal fibrosis [2]. Blood vessels mayparticipate in the genesis of IPF, and similar disruptions in cytokines have been described in patients withIPF and PH. The latter suggests that these disorders share pathogenic features and that one may influence,generate or perpetuate the other. Different cellular processes have been described during pulmonary arteryremodelling of PH-associated IPF, including endothelial dysfunction [3], the endothelial-to-mesenchymaltransition as a source of myofibroblasts [4], as well as pulmonary artery smooth muscle proliferation andtransition to myofibroblasts [5].

Although PH is clearly associated with worse IPF outcomes, it remains unproven that treating PH withvasoactive therapies results in improvement. Although effective in patients with PH, endothelin-1 (ET-1)receptor antagonists failed in IPF clinical trials. Deleterious effects of vasodilators in hypoxic PH, such asPH-associated IPF, are attributed to increased oxygen desaturation mediated by the propensity to worsenventilation/perfusion (V′/Q′) matching by dilating vessels that are perfusing poorly ventilated lung units [6].In this regard, the phosphodiesterase type 5 (PDE5) inhibitor sildenafil improved pulmonaryhaemodynamics by maintaining V′/Q′ ratios in the short term in a cohort of patients with PH secondary tolung fibrosis, suggesting a favourable profile of sildenafil for treating IPF [7]. STEP-IPF trial investigatorstreated patients with severe IPF (diffusing capacity of the lung for carbon monoxide (DLCO) <35%) withsildenafil. Sildenafil did not meet the primary outcome measure of an improved 6MWD compared withplacebo in the overall study population; however, small but significant improvements were observed insecondary measures, including improved DLCO, arterial oxygenation, dyspnoea symptoms and quality of lifecompared with placebo [8]. Sildenafil significantly improved 6MWD (by 99.3 m) in a subgroup of patientswith IPF and right-sided ventricular systolic dysfunction that was associated with improved quality of life.Sildenafil may have acted in this group by addressing PH [9]. Sildenafil ameliorates right ventriclehypertrophy and fibrosis, as well as pulmonary vascular remodelling, in animal models ofbleomycin-induced pulmonary fibrosis associated with PH by improving nitric oxide synthase coupling andreducing reactive oxygen species signalling [10, 11]. Sildenafil also reduces endothelial dysfunction andinhibits human pulmonary artery smooth muscle cell (HPASMC) proliferation in cell models [12, 13].

Unfortunately, there are no approved targeted therapies for PH in patients with IPF. Although growingevidence suggests beneficial effects of sildenafil on PH associated with IPF, the direct effects of sildenafilon the pulmonary arteries of patients with PH-associated IPF must be determined. In this study, weanalysed the vasodilator and anti-remodelling properties of sildenafil ex vivo in the pulmonary arteries ofpatients with PH associated with IPF. Furthermore, we evaluated the effect of sildenafil on the V′/Q′ ratioin a bleomycin animal model.

Materials and methodsA more detailed version of the methods outlined below can be found in the online supplementary material.

PatientsPulmonary arteries were obtained from: 1) patients with PH-associated IPF (n=8); 2) patients with IPFwithout PH (n=9); 3) patients with PH without IPF (n=4); and 4) donor subjects without any lung disease(n=18). This protocol was approved by the local research and independent ethics committee of theUniversity General Consortium Hospital of Valencia (CEIC26/2013). Written informed consent wasobtained from each participant. Please refer to the online supplementary material for further details.

Preparation of pulmonary artery rings for functional studiesPulmonary arteries were isolated and prepared as outlined previously [14]. Isometric tension of the arterialrings was recorded as we reported previously [14]. We employed two different protocols. 1) Relaxantprotocol: cumulative concentrations of sildenafil were added to pre-contracted arteries following 1 µMserotonin (5-HT) treatment. The results are expressed as the percentage of sildenafil relaxation comparedwith maximal relaxation produced by 0.1 mM papaverine. 2) Contraction protocol: sildenafil (0.1–10 µM)was added to the organ bath 30 min before cumulative concentrations of 5-HT (0.1 nM to 10 µM) wereadded. Details are described in the online supplementary material.

1738 DOI: 10.1183/13993003.01259-2015

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

Real-time RT-PCR analysisTotal RNA was obtained from the pulmonary arteries of patients from different groups. The relativequantification of different transcripts was determined using the 2−ΔΔCt method with glyceraldehydephosphate dehydrogenase as the endogenous control and normalised to a control group, as describedpreviously [15]. Details are described in the online supplementary material.

Immunofluorescence and Western blottingCollagen type I (Col I), α-smooth muscle actin (α-SMA), phospho-extracellular regulated kinases 1 and 2(ERK1/2), phospho-SMAD3, S100A4, and PDE5 were detected in human lung tissue or in pulmonaryartery rings by immunofluorescence or Western blot as outlined previously [16]. Details are described inthe online supplementary material.

Animal studiesAnimal experimentation and handling were performed in accordance with the guidelines of the committeeof animal ethics and well-being of the University of Valencia (Valencia, Spain). A single dose of 3.75 U·kg−1

bleomycin was administered intratracheally [16]. In both the sham control and bleomycin groups, a singledose of 1 mg·kg−1 sildenafil was administered intraperitoneally on day 21. The V′/Q′ ratio was evaluated 2 hafter administration using small-animal computed tomography (micro-CT) and single-photon emissioncomputed tomography (SPECT) (Oncovision® micro-CT-SPECT-PET Imaging System; Albira, Valencia,Spain) imaging with the radioisotopes diethylene-triamine-pentaacetate (DTPA)-99mTc for ventilation(10 mCi) and microaggregated albumin (MAA)-99mTc for perfusion (0.5–1 mCi), as outlined previouslywith modifications [17]. Details are described in the online supplementary material.

Statistical analysisThe Kruskal–Wallis test followed by Dunn’s post hoc tests were used as nonparametric tests when morethan two groups were compared in the human studies. The Mann–Whitney U-test was employed tocompare two groups. Two-tailed paired t-tests were used to compare two groups of dependent samples inthe animal and cell studies, and unpaired t-tests were used for independent samples. Multiple comparisonswere analysed by a one- or two-way ANOVA followed by Bonferroni post hoc tests. Details are describedin the online supplementary material.

ResultsRelaxant and anti-contractile effects of sildenafil on pulmonary arteries from patients with IPFFour different groups based on the clinical characteristics defined in table 1 were used in the experiments.Sildenafil induced concentration-dependent relaxation of pulmonary artery rings that was greater in thecontrol and IPF groups (maximum relaxation effect of 41.3±11.6% and 37.22±15%, respectively) than thatthe PH+IPF and PH groups (9.8±5.8% and 9.46±4.23%; p<0.05 compared with the control and IPF groups;figure 1a–d). When the endothelium was removed, the maximal relaxation effect of sildenafil decreased inthe control and IPF pulmonary artery rings to 20.8±10% and 20.19±10.5%, respectively, whereas themaximal relaxant effect of sildenafil remained similar in the PH+IPF and PH groups (figure 1c and d).

In other experiments, pulmonary artery rings were incubated with sildenafil (0.1–10 μM) 30 min beforeadministration of cumulative doses of 5-HT (0.1 nM to 10 µM). Sildenafil did not significantly inhibit5-HT-induced contractions in control donors (figure 2a). In contrast, 5-HT-induced contraction wassignificantly inhibited by sildenafil in a concentration-dependent manner in the IPF (maximal inhibition,54.3±3%; p<0.05 versus control; figure 2b), PH+IPF (maximal inhibition, 68.3±4%; p<0.05 versus control;figure 2c), and PH arterial rings (maximal inhibition, 73.6%; p<0.05 versus control; figure 1d).Importantly, the inhibitory effect of sildenafil on 5-HT-induced contraction was significantly higher in thePH+IPF and PH arterial rings than those from patients with IPF (p<0.05).

Characterisation of remodelling markers in pulmonary arteries from controls and patients withIPF with or without PHThe pulmonary artery functional experiment results indicated that sildenafil had different relaxant/anti-contractile effects in pulmonary artery from control, IPF, and PH+IPF, which may be explained bydifferences in pulmonary artery remodelling components. Thus, mesenchymal/myofibroblast markers werestudied in pulmonary arteries. α-SMA, vimentin, Col I, 5-HT receptor 2A and transforming growth factor(TGF)-β1 expression were significantly upregulated in pulmonary arteries from patients with IPF and PH+IPF compared with those from controls, suggesting increased muscularisation, extracellular matrixdeposition, and fibrosis (figure 3a). Furthermore, vimentin, Col I, 5-HT receptor 2A and TGF-β1expression were higher in pulmonary arteries from patients with PH+IPF when compared with those frompatients with IPF (figure 3a), which indicates their role in PH development. The endothelial markers

DOI: 10.1183/13993003.01259-2015 1739

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

TABLE 1 Clinical characteristics

Control donor subjects IPF patients PH-associated IPF PH

Subjects n 18 9 8 4Age years 59 (42–71) 61 (56–75) 65(52–73) 45 (42–52)Males/females 15/3 12/5 25/11 3/1SmokingNever smoked/smokers 7/11 3/6 4/4 4/0Pack-years 26 (0–32) 28.3 (6–35) 31 (9–38)

FEV1 % pred ND 72.8 (58–101) 73 (53–98) 92 (91–95)FVC % pred ND 70.2 (63–79) 71 (48–76) NDTLC % pred ND 73.5 (45–89) 66 (43–90) NDDLCO % pred ND 50.1 (34–61) 40.8 (20–61) NDGround glass# % 0 19 (8–39) 22 (9–35) 0Honeycombing¶ % 0 28 (15–40) 26 (12–39) 0PaO2 mmHg 94 (88–96) 65 (45–88) 60 (40–85) 62 (48–75)mPAP mmHg·L−1·min−1 ND 20.5 (16–22) 43 (36–48) 69 (42–74)NAC+ (yes/no) 0/21 4/5 5/4 0/4Pirfenidone+ (yes/no) 0/21 2/7 1/7 0/4

Data are presented as medians (interquartile range), unless otherwise stated. IPF: idiopathic pulmonaryfibrosis; PH: pulmonary hypertension; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity;TLC: total lung capacity; DLCO: diffusion capacity of the lung for carbon monoxide; PaO2: arterial blood oxygentension; mPAP: mean pulmonary artery pressure; NAC: N-acetyl-L-cysteine; ND: not determined. #: percentageof pulmonary parenchyma with ground glass on a computed tomography (CT) image; ¶: percentage ofpulmonary parenchyma with honeycombing on a CT image; +: patients who received this treatment at the timeof pulmonary biopsy.

0

20

40

60

–10 –9

*

**

*

*

*

*

**

–8

log Sildenafil M

Control pulmonary

artery

a)

–7 –6 –5

E (+)

E (–)

Re

laxa

tio

n %

of

pa

pa

veri

ne

0.1

mM

0

20

40

60

–10 –9 –8

log Sildenafil M

IPF pulmonary arteryb)

–7 –6 –5

E (+)

E (–)

Re

laxa

tio

n %

of

pa

pa

veri

ne

0.1

mM

E (+)

E (–)0

20

40

60

–10 –9 –8

log Sildenafil M

IPF + PH pulmonary

artery

c)

–7 –6 –5

## E (+)

E (–)

Re

laxa

tio

n %

of

pa

pa

veri

ne

0.1

mM

0

20

40

60

–10 –9 –8

log Sildenafil M

PH pulmonary arteryd)

–7 –6

##

#

–5

Re

laxa

tio

n %

of

pa

pa

veri

ne

0.1

mM

FIGURE 1 Relaxant effects of sildenafil on pulmonary arteries. Concentration-dependent relaxant curves ofsildenafil on pulmonary arteries pre-contracted with 1 µM serotonin from control donors (n=10) and patientswith idiopathic pulmonary fibrosis (IPF) (n=6), IPF plus pulmonary hypertension (IPF+PH) (n=6) and PH (n=4).Four arterial rings per patient were used. The pulmonary artery endothelium (E) was removed for someexperiments to analyse the effect of endothelium on sildenafil relaxant activity. The results are shown asmean±SE. Two-way ANOVA followed by Bonferroni post hoc tests. *: p<0.05 versus pulmonary arteries withoutendothelium; #: p<0.05 compared with baseline tension.

1740 DOI: 10.1183/13993003.01259-2015

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

endothelial nitric oxide synthase (eNOS), vascular endothelial (VE)-cadherin, vascular endothelial growthfactor receptor (VEGFR) and factor VIII (FVIII) were downregulated in pulmonary arteries from patientswith IPF and PH+IPF (figure 3a), indicating impaired endothelial function or a change in endothelialphenotype. Phosphorylation of ERK1/2 and SMAD3 was greater in pulmonary arteries from patients withPH+IPF; however, this phosphorylation was less in pulmonary arteries from patients with IPF comparedwith controls (figure 3b and c; p<0.05). PDE5 expression was upregulated in pulmonary arteries frompatients with IPF and PH+IPF (figure 3a and c) and mainly located in endothelial cells and the intimallayer (figure 3c), whereas PDE5 was weakly expressed in pulmonary arteries from control donors.

Anti-remodelling effects of sildenafil on human pulmonary arteries from patients with IPFIn other ex vivo experiments (experimental design details can be found in the online supplementarymaterial), pulmonary artery ring explants were cultured and stimulated with 5 ng·mL−1 TGF-β1 for 72 h inthe presence or absence of sildenafil (10 nM–1 μM). TGF-β1 (5 ng·mL−1) treatment resulted in decreasedexpression of the endothelial markers VE-cadherin and eNOS. In contrast, this treatment increased theexpression of PDE5 and the mesenchymal markers Col I and vimentin, but not α-SMA or TGF-β1 inpulmonary artery rings from control subjects (figure 4a–c). These changes were also observed in pulmonaryarteries from patients with IPF, where the loss in endothelial and the increase in mesenchymal markerswere higher, including significant upregulation of α-SMA and TGF-β1 (figure 4d–f ). Sildenafil inhibitedthe effect of TGF-β1 on the loss of endothelial markers and upregulation of the pulmonary arteryremodelling markers in pulmonary artery rings of the control and IPF groups at 10–100 nM, suggestingpulmonary artery anti-remodelling properties in patients with IPF.

Sildenafil inhibited TGF-β1-induced pulmonary artery endothelial-to-mesenchymal transition andthe transition of smooth muscle cells to myofibroblastsSubsequent experiments were designed to explore the anti-remodelling effects of sildenafil on isolated andcultured human pulmonary artery endothelial cells (HPAECs) and HPASMCs from patients with IPF.Incubating the HPAECs with TGF-β1 changed their endothelial phenotype to a mesenchymal/myofibroblast

0

20

40

60

80

100

–10 –9 –8

log 5-HT M

Control pulmonary arterya)

–7 –6 –5

Control

Sildenafil 0.1 µM

Sildenafil 1 µM

Sildenafil 10 µM

Re

laxa

tio

n %

of

pa

pa

veri

ne

0.1

mM

0

20

40

60

80

100

–10 –9 –8

log 5-HT M

IPF pulmonary arteryb)

–7 –6 –5

*#

**

**

*

Control

Sildenafil 0.1 µM

Sildenafil 1 µM

Sildenafil 10 µM

Co

ntr

acti

on

% o

f

KC

I 8

0 m

M0

20

40

60

80

100

–10 –9 –8

log 5-HT M

IPF + PH

pulmonary artery

c)

–7 –6 –5

*

**

*

*

*

*

**

*

**

**

Control

Sildenafil 0.1 µM

Sildenafil 1 µM

Sildenafil 10 µM

Re

laxa

tio

n %

of

pa

pa

veri

ne

0.1

mM

0

20

40

60

80

100

–10 –9 –8

log 5-HT M

PH pulmonary arteryd)

–7 –6 –5

Control

Sildenafil 0.1 µM

Sildenafil 1 µM

Sildenafil 10 µM

Co

ntr

acti

on

% o

f

KC

I 8

0 m

M

FIGURE 2 Effect of sildenafil on serotonin (5-HT)-induced pulmonary contraction. Pulmonary arteries from control donors (n=12) and patients withidiopathic pulmonary fibrosis (IPF) (n=6), IPF plus pulmonary hypertension (IPF+PH) (n=6) and PH (n=4) were incubated with sildenafil for 30 minfollowed by increasing 5-HT concentrations. Four arterial rings per patient were used. The results are shown as mean±SE. Two-way ANOVAfollowed by Bonferroni post hoc tests. *: p<0.05 compared with control donor; #: p<0.05 compared with the PH+IPF and PH groups.

DOI: 10.1183/13993003.01259-2015 1741

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

phenotype characterised by loss of the endothelial markers VE-cadherin, VEGFR1, FVIII and eNOS, and anincrease in the mesenchymal markers Col I, α-SMA and vimentin, as well as the expression of the profibroticfactors TGF-β1 and PDE5 (figure 5a). Sildenafil prevented these changes at a low concentration (10 nM) andmaintained the endothelial phenotype. TGF-β1 increased the expression of Col I and vimentin in HPASMCsin a concentration-dependent manner; this effect was inhibited by sildenafil (figure 5b). Furthermore,sildenafil inhibited TGF-β1-induced HPASMC proliferation (figure 5c). Adding 5 ng·mL−1 TGF-β1 toHPAECs and HPASMCs from patients with IPF increased the phosphorylation of ERK1/2 and SMAD3,which was inhibited by sildenafil (figure 6a and b). In parallel experiments, stimulating HPAECs andHPASMCs with 5 ng·mL−1 TGF-β1 enhanced expression of Col I and the S100A4 fibrotic marker, whichwere inhibited by sildenafil, PD98059 (ERK1/2 inhibitor) and SIS3 (SMAD3 inhibitor). These results

a)

0

2

4

6p=0.0089

8

10

α-S

MA

/GA

PD

H

mR

NA

exp

ressio

n

p=0.0079

ControlIPFIPF+PH

0

5

10

15

p=0.0159

20

Vim

en

tin

/GA

PD

H

mR

NA

exp

ressio

n

p=0.0285

p=0.0070

0

5

10

15p=0.0023

20

Co

l ty

pe

I/G

AP

DH

mR

NA

exp

ressio

n

p=0.0286

p=0.0010

0

1

2

3

4

p=0.0952

5

TG

F-β

1/G

AP

DH

mR

NA

exp

ressio

n

p=0.0286p=0.0079

0

1

2

3

4

p=0.01215

6

PD

E5

/GA

PD

H

mR

NA

exp

ressio

n

p=0.0019

0

2

4

6

p=0.0286

8

5-H

TR

2A

/GA

PD

H

mR

NA

exp

ressio

n

p=0.0039

p=0.0210

0Control

p-ERK 1

p-ERK 2

IPF

** *

*

IPF+PH

Controlb) IPF IPF+PH

p-ERK1/2

ERK1/2

1

2

3

p-E

RK

1/2

/to

tal

ER

K1

/2

pro

tein

exp

ressio

n

0

0.5

1.0

1.5

2.0

2.5p=0.1429

eN

OS

/GA

PD

H

mR

NA

exp

ressio

n

p=0.0159

0

0.5

1.0

1.5p=0.0091

VE

-ca

dh

eri

n/G

AP

DH

mR

NA

exp

ressio

n

p=0.0076

0

1

2

3p=0.0132

VE

GF

R-1

/GA

PD

H

mR

NA

exp

ressio

n

p=0.0065

0

0.5

1.0

1.5

p=0.00112.0

FV

III/

GA

PD

H

mR

NA

exp

ressio

n

p=0.0099

0Control IPF IPF+PH

Control IPF IPF+PH Control IPF IPF+PH

p-SMAD3

c)

SMAD3

0.5

1.0

1.5

2.0

2.5

p-S

MA

D3

/to

tal

SM

AD

3

pro

tein

exp

ressio

n

FIGURE 3 Basal expression of pulmonary artery remodelling and signalling markers. Pulmonary arteries isolated from control donors (n=18) andpatients with idiopathic pulmonary fibrosis (IPF) (n=9) and IPF plus pulmonary hypertension (IPF+PH) (n=8) were collected to measure: a) genemRNA expression of remodelling and endothelial markers; protein expression of b) phospho-extracellular regulated kinases 1 and 2 (ERK1/2) andphospho-SMAD3 by Western blot; and c) phosphodiesterase type 5 (PDE5) and alpha smooth muscle actin (α-SMA) by immunofluorescence (DAPI:4’,6-diamidino-2-phenylindole). a) The data are presented as box and whisker plots with median, interquartile range and range values. Exactp-values were obtained using the Kruskal−Wallis test and Dunn’s post hoc tests. b) The data are expressed as a ratio of total ERK1/2 and SMAD3,and normalised to the control donor group. Representative Western blots of p-ERK1/2 and p-SMAD3 in pulmonary arteries. One-way ANOVAfollowed by Bonferroni post hoc tests. *: p<0.05 compared with the control donor. c) Representative immunofluorescence of lung tissue indifferent patient groups. Scale bar=200 µm. The immunoglobulin G isotype control was negative (data not shown). GAPDH: glyceraldehyde3-phosphate dehydrogenase; Col type I: collagen type I; TGF-β1: transforming growth factor-β1; VEGFR: vascular endothelial growth factorreceptor; FVIII: factor VIII.

1742 DOI: 10.1183/13993003.01259-2015

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

indicate that sildenafil partially mediates anti-remodelling effects on human pulmonary arteries frompatients with IPF by inhibiting ERK1/2 and SMAD3.

Sildenafil did not affect V′/Q′ mismatch in a rat model of bleomycin-induced pulmonary fibrosisassociated with PHRats were instilled intratracheally with saline (control) or bleomycin on day 1 to induce pulmonary fibrosisand PH on day 21 as we outlined previously [16]. The V′/Q′ study began 2 h after administering 1 mg·kg−1

sildenafil intraperitoneally. Ventilation images were taken after breathing DTPA-99mTc, and perfusionimages were taken after perfusing MAA-99mTc. As expected, the V′/Q′ ratio was clearly impaired in thelungs of bleomycin-treated rats compared with that in control animals. Administering sildenafil on day 21(once pulmonary fibrosis and PH were established) increased the perfusion signal slightly (figure 7b), butdid not modify the V′/Q′ ratio (figure 7c).

DiscussionNo specific therapy currently exists for PH associated with lung diseases, such as IPF. According to recentguidelines, vasodilators approved for pulmonary artery hypertension (PAH) are not recommended forpatients with PH due to lung disease [18, 19]. However, a subset of patients with severe PH related to theirlung disease, namely out-of-proportion PH, can be treated according to the recommendations for PAH[18]. The incompletely understood and likely complex pathogenesis, together with the small series attesting

Basalc)

Control

patients

Control

patients

IPF

patients

IPF

patients

TGF-β1 + Sil 1 µMTGF-β1 5 ng·mL–1

Basalf) TGF-β 1 + Sil 1 µMTGF-β1 5 ng·mL–1

0

VE

-ca

dh

eri

nControlTGF-β1 5ng·mL–1

Sildenafil 1µM

* *

#

#

eN

OS

0.5

1.0

1.5

2.0

a)

mRN

A ex

pres

sion

0C

ol

typ

e I

ControlTGF-β1 5ng·mL–1

Sildenafil 1µM

*

*# *# *#

**

0.5

1.0

1.5

2.0

b)

mRN

A ex

pres

sion

Vim

en

tin

α-S

MA

TG

F-β

1

PD

E5

Co

l ty

pe

I

Vim

en

tin

α-S

MA

TG

F-β

1

PD

E5

0

VE

-ca

dh

eri

n

ControlTGF-β1 5 ng·mL–1

Sildenafil 10 nM

*

*

*

* *

*#

*#

eN

OS

0.5

1.0

1.5

*

*#*#

* * *

#

d)

mRN

A ex

pres

sion

0

ControlTGF-β1 5 ng·mL–1

Sildenafil 10 nM

0.5

1.0

1.5

e)

mRN

A ex

pres

sion

Sildenafil 100 nMSildenafil 1 µM

Sildenafil 100 nMSildenafil 1 µM

*#

*#

*#

FIGURE 4 Effect of sildenafil on transforming growth factor (TGF)-β1-induced ex vivo pulmonary artery ring remodelling. Pulmonary artery ringsfrom a–c) donor controls (n=4) and d–f ) patients with idiopathic pulmonary fibrosis (IPF) (n=4) were incubated with sildenafil (10 nM to 10 µM) for1 h and then stimulated with 5 ng·mL−1 TGF-β1 for 72 h. Artery rings were homogenised to extract RNA for gene expression analysis or embeddedin paraffin for immunofluorescence analysis (DAPI: 4’,6-diamidino-2-phenylindol) as shown in e) and f). Scale bar=150 μm. White arrows indicateadventitial and smooth muscle layers. Yellow arrows indicate endothelial cells. Four arterial rings per patient were used. VE: vascular endothelial;eNOS: endothelial nitric oxide synthase; Col type 1: collagen type 1; α-SMA: alpha smooth muscle actin; PDE5: phosphodiesterase type 5;Sil: sildenafil; COL 1: collagen type 1. The results are shown as mean±SE. Two-way ANOVA followed by Bonferroni post hoc tests. *: p<0.05compared with control; #: p<0.05 compared with TGF-β1.

DOI: 10.1183/13993003.01259-2015 1743

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

to the utility of treating PH in patients with IPF, provide a foundation and basis for further consideration ofrandomised controlled trials of pulmonary vasoactive therapies for this patient phenotype. In this study, weanalysed the effects of sildenafil on relaxation and anti-contractile and anti-remodelling properties in an exvivo/in vitro system of pulmonary arteries from patients with IPF with and without severe PH. Our findings

0

Co

ntr

ol

*

#

#

0.5

1.0

1.5

TGF-β1

5 ng·mL–1

a)

VE-c

adhe

rin/G

APDH

mRN

A ex

pres

sion

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*#

#

0.5

1.0

2.5

1.5

2.0

TGF-β1

5 ng·mL–1

Vim

entin

/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

**

1

2

5

3

4

TGF-β1

5 ng·mL–1

Vim

entin

/GAP

DHm

RNA

expr

essi

on

TG

F-β

1 1

ng

·mL

–1

TG

F-β

1 5

ng

·mL

–1

TG

F-β

1 1

0 n

g·m

L–

1

0

Co

ntr

ol

*

#*

#*#*

1

280

120

160

200

TGF-β1

5 ng·mL–1

Col t

ype

I/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

#

#

#

0.5

1.0

2.0

1.5

TGF-β1

5 ng·mL–1

VEGF

R1/G

APDH

mRN

A ex

pres

sion

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

#*#*

#

1

2

3

TGF-β1

5 ng·mL–1

Col t

ype

I/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

#

#

#

0.5

1.0

2.0

1.5

TGF-β1

5 ng·mL–1

FVIII

/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

#*

*

#

1

2

4

3

TGF-β1

5 ng·mL–1

Vim

entin

/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

*#*#

#

0.5

1.0

2.0

1.5

TGF-β1

5 ng·mL–1TG

F-β1

/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

##

#

0.5

1.0

1.5

TGF-β1

5 ng·mL–1

e-N

OS/G

APDH

mRN

A ex

pres

sion

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

*#*

*#

1

2

3

c)TGF-β1

5 ng·mL–1

BrdU

inco

rpor

atio

n x-

fold

of c

ontr

ol

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

#

#

0.5

1.0

2.5

2.0

1.5

TGF-β1

5 ng·mL–1

PDE5

/GAP

DHm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

##

#

0.5

1.0

2.0

2.5

1.5

TGF-β1

5 ng·mL–1

α-S

MA/

GAPD

Hm

RNA

expr

essi

on

TG

F-β

1

Sil

de

na

fil

10

nM

Sil

de

na

fil

10

0 n

M

Sil

de

na

fil

1 µ

M

0

Co

ntr

ol

*

**

1

2

3

b)TGF-β1

5 ng·mL–1

Col t

ype

I/GAP

DHm

RNA

expr

essi

on

TG

F-β

1 5

ng

·mL

–1

TG

F-β

1 1

0 n

g·m

L–

1

TG

F-β

1 1

ng

·mL

–1

FIGURE 5 Effects of sildenafil on transforming growth factor (TGF)-β1-induced endothelial- and smooth muscle cell-mesenchymal transition.a) Human pulmonary artery endothelial cells and b) human pulmonary artery smooth muscle cells (HPASMCs) were isolated from pulmonaryarteries of patients with idiopathic pulmonary fibrosis (n=4). The cells were incubated with sildenafil (10 nM to 1 µM) for 30 min and then stimulatedwith 5 ng·mL−1 TGF-β1 for a) 72 h or b) 48 h. Endothelial and mesenchymal marker gene mRNA expression was analysed. c) HPASMCs from patientswith IPF were incubated with sildenafil for 30 min and then stimulated with 5 ng·mL−1 TGF-β1 for 48 h in 96-well plates to measure cell proliferationwith the bromodeoxyuridine (BrdU) assay. The results are shown as mean±SE of n=4 cell populations from patients with IPF. VE: vascular endothelial;GAPDH: glyceraldehyde 3-phosphate dehydrogenase; VEGFR: vascular endothelial growth factor receptor; FVIII: factor VIII; eNOS: endothelial nitricoxide synthase; α-SMA: alpha smooth muscle actin; Col type1: collagen type 1; α-SMA: alpha smooth muscle actin; PDE5: phosphodiesterase type 5.Two-way ANOVA followed by Bonferroni post hoc tests. *: p<0.05 compared with control; #: p<0.05 compared with TGF-β1.

1744 DOI: 10.1183/13993003.01259-2015

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

may add scientific value to support treatment of out-of-proportion PH associated with the IPF phenotypeusing PDE5 inhibitors.

Clinical studies have reported that sildenafil (50 mg orally) causes acute pulmonary vasodilation in patientswith severe IPF and PH (mean pulmonary arterial pressure, 42 mmHg), mainly in well-ventilated areaswith available NO, without affecting hypoxic vasoconstriction or oxygen saturation, and thus withoutdisturbing the V′/Q′ match [7]. The long-term STEP-IPF trial demonstrated that sildenafil improved6MWD in a subset of patients with severe IPF and right-sided ventricular systolic dysfunction measuredby echocardiography [9]. These positive results corroborate previous findings in small cohorts of patientswith PH-associated with severe IPF [20, 21] but not in patients with IPF and mild to moderate PH [22].

In this study, we observed a direct vasodilatory effect of sildenafil in 5-HT-precontracted pulmonary arteriesfrom healthy control patients and from patients with IPF, which was partially endothelium-dependent (40%with endothelium versus 20% maximal relaxation without endothelium). A well-known vasodilatorymechanism of sildenafil implicates the production of NO from eNOS in endothelial cells. The endothelialrelease of NO activates guanylate cyclase (GC) in pulmonary artery smooth muscle cells to produce cyclicguanosine monophosphate (cGMP) and protein kinase G (PKG), which induce vasodilation [23]. However,an endothelial-independent vasodilatory effect of sildenafil was also described. For example, sildenafilinduces endothelial-independent relaxation in vessels of humans [24] and piglets [25], as well as rat [26]pulmonary arteries. Moreover, sildenafil significantly reduced acute hypoxic pulmonary vasoconstriction inperfused lungs of eNOS-deficient mice, indicating that the effects of sildenafil occur even when eNOS isimpaired [27]. We observed a small degree of vasodilation in 5-HT-precontracted pulmonary arteries frompatients with PH+IPF and PH that was independent of the presence of endothelium, suggesting that theeNOS-NO endothelial axis is impaired in patients with PH+IPF, as described previously [28]. Synthesis ofcGMP also occurs in response to natriuretic peptides after activation of particulate GC in pulmonaryvascular smooth muscle cells [29]. Serum natriuretic peptides are elevated as a compensatory mechanism inpatients with PH-associated IPF [30], which partially explains the increased pulmonary vasodilation after asingle oral administration of sildenafil in patients with PH+IPF [7].

In our study, the small degree of vasodilation observed in our ex vivo system of pulmonary arteries frompatients with PH+IPF could be attributed to the lack of circulating natriuretic peptides and the lack ofNO-enriched lung tissue in well-ventilated areas where sildenafil induced a selective increase in vasodilation[7]. This is the rationale for the recent synergism observed between the natriuretic peptide system andsildenafil in an animal model of lung fibrosis associated with PH [31]. Although we did not explore the roleof natriuretic peptides in this study, we speculate that small local pulmonary artery production of natriuretic

p-ERK1/2

b)

Co

ntr

ol

Sil

10

nM

Sil

10

0 n

M

Sil

1 µ

M

PD

98

05

9 1

0 µ

M

SIS

3 1

0 µ

M

TG

F-β

1

ERK1/2

p-SMAD3

SMAD3

TGF-β1 5 ng·mL–1 c)

Co

ntr

ol

Sil

10

nM

Sil

10

0 n

M

Sil

1 µ

M

PD

98

05

9 1

0 µ

M

SIS

3 1

0 µ

M

TG

F-β

1

TGF-β1 5 ng·mL–1

Col type I

S100A4

β-actin

d)

Co

ntr

ol

Sil

10

nM

Sil

10

0 n

M

Sil

1 µ

M

PD

98

05

9 1

0 µ

M

SIS

3 1

0 µ

M

TG

F-β

1

TGF-β1 5 ng·mL–1

Co

ntr

ol

Sil

10

nM

Sil

10

0 n

M

Sil

1 µ

M

PD

98

05

9 1

0 µ

M

SIS

3 1

0 µ

M

TG

F-β

1TGF-β1 5 ng·mL–1a)

0

1

p-ERK1/2/

ERK1/2

p-SMAD3/

SMAD3

2

X-f

old

of

co

ntr

ol

3

*

** *

* *

#

##

#*#

#

* *

**

#*

#*# #

# # ##

0

1

p-ERK1/2/

ERK1/2

p-SMAD3/

SMAD3

2

X-f

old

of

co

ntr

ol

3

#*#*

# # ## # #

*

**

*

0

1

Col type I S100A4

2

X-f

old

of

co

ntr

ol

3

4

**

**

#* #*#

# # # ##

Col type I S100A40

1

2

X-f

old

of

co

ntr

ol

3

4

FIGURE 6 Sildenafil inhibits the endothelial- and smooth muscle-to-mesenchymal transition by inhibiting phosphorylation of extracellularregulated kinases 1 and 2 (ERK1/2) and SMAD3. a, c) Pulmonary artery endothelial cells and b, d) artery smooth muscle cells were incubated withsildenafil (Sil) (10 nM to 1 µM), PD98059 (ERK1/2 inhibitor), or SIS3 (SMAD3 inhibitor) for 30 min and then stimulated with 5 ng·mL−1 transforminggrowth factor (TGF)-β1 for 30 min (a, b) or 72 h (c, d). Protein expression of phospho-ERK1/2 and phospho-SMAD3 (a, b), collagen type I (Coltype I) and S100A4 (c, d) were measured by Western blot. The data are expressed as a ratio of total ERK1/2 and SMAD3 (a, b) or total β-actin (c, d)and normalised to the control group. Representative Western blots are shown. The results are shown as mean±SE (n=4). Two-way ANOVA followedby Bonferroni post hoc tests. *: p<0.05 compared with control; #: p<0.05 compared with TGF-β1.

DOI: 10.1183/13993003.01259-2015 1745

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

peptides may be partially responsible for the actions of sildenafil that we observed. Similar selectivevasodilator properties were observed after acute administration of sildenafil in the bleomycin-inducedpulmonary fibrosis animal model associated with PH, in which the V′/Q′ ratio remained unaltered.

V′/Q′ SPECT imaging is a well-established nuclear medicine technique that provides spatial information ofrespiratory gas exchange, ventilation of alveolar units and perfusion of the pulmonary capillary beds.Chronic pulmonary hypoxic diseases such as chronic obstructive pulmonary disease (COPD) or IPF havean impaired ventilation and perfusion of the lung units and can be monitored by the analysis of theinhaled DTPA-99mTc signal and perfused MAA-99mTc. However the use of this technique in preclinicalstudies is limited. To our knowledge there is only one study that observed an impaired V′/Q′ SPECT ratioin a rat model of COPD smoking between the 8th to 24th weeks of smoking [17]. In this work weobserved an impaired V′/Q′ SPECT ratio at the end of the 21-day bleomycin-induced lung fibrosisprocedure. Sildenafil weakly increased perfusion signal without affecting V′/Q′ ratio, confirming previousresults in humans [7] and the utility of this technique to the preclinical in vivo study of vasodilators inhypoxic lung disorders such as IPF. However, the selective actions of sildenafil on well-ventilated areasshould be demonstrated with repeated administration of sildenafil in an animal therapeutic protocol ratherthan in acute experiments, which is a limitation of this study and warrants future experiments.

Different results were observed for sildenafil in the anti-contractile protocol. Chronic administration ofsildenafil is the optimal scenario in patients with severe PH+IPF; thus, sustained inhibition of PDE5prevents the contractile effects of elevated vasoactive mediators, such as ET-1 and 5-HT, among others. Inthe present study, pre-incubation with 10 μM sildenafil minimally affected the 5-HTconcentration-response curves (10% inhibition) in healthy PAs as shown previously by RIED et al. [32]using ET-1 as a vasoactive mediator [32]. In contrast to our results observed for healthy donors, 10 μMsildenafil resulted in nearly 50% inhibition of 5-HT-induced contraction in pulmonary arteries frompatients with IPF; however, the effect of sildenafil was greater in pulmonary arteries from patients with

0

Co

ntr

ol

BL

M

BL

M+

SIL

SIL

d) Control BLM BLM+SIL

V'

Q'

0.2

0.4

V' D

TP

A-9

9m

Tc

co

rre

cte

d i

nte

nsit

y

0.6

0.8a)

**

0

Co

ntr

ol

BL

M

BL

M+

SIL

SIL

0.2

0.4

Q' M

AA

-99

mTc

co

rre

cte

d i

nte

nsit

y

0.6

0.8b)

*

#

0

Co

ntr

ol

BL

M

BL

M+

SIL

SIL

0.5

1.0

V'/Q

'

1.5c)

**

FIGURE 7 Sildenafil does not modify ventilation/perfusion (V′/Q′) mismatch in a rat model of pulmonary fibrosisassociated with bleomycin (BLM)-induced pulmonary hypertension. A single 3.75 U·kg−1 dose of BLM or vehiclewas administered intratracheally on day 0. A single 1 mg·kg−1 dose of sildenafil (SIL) was administeredintraperitoneally after 21 days. The animals were ventilated on a rodent ventilator with 10 mCidiethylene-triamine-pentaacetate (DTPA)-99mTc for 15 min, 2 h after sildenafil administration. After DTPA-99mTcdelivery, the animals were removed from the ventilator and allowed to breathe freely. Single-photon emissioncomputed tomography (SPECT) scans were acquired on an ALBIRA micro-CT-PET-SPECT system (Oncovision).After the SPECT scan, the rats were injected with 0.5–1 mCi microaggregated albumin (MAA)-99mTc via the tailvein. Perfusion imaging entailed another SPECT scan. The relationship between the ventilation and perfusiondata was determined using PMOD™ software to analyse the intensity of radiation (arbitrary units) of each volumeof interest. a–c) Mean±SE of the radiation intensity of the V′ signal, Q′ signal, and their ratio. d) Representativeventilation and perfusion images. Results were analysed in 10 animals per experimental condition. One-wayANOVA followed by Bonferroni post hoc tests. *: p<0.05 compared with control; #: p<0.05 compared with BLM.

1746 DOI: 10.1183/13993003.01259-2015

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

PH+IPF and PH. Differences in the anti-contractile effects of sildenafil can be partially explained by thedifferent PDE5 expression levels among patients: PDE5 was overexpressed in pulmonary arteries frompatients with PH+IPF (and to a lesser extent in patients with IPF) compared with that in healthy subjects.However, we cannot exclude that other mechanisms may be involved.

We selected 5-HT as a vasoactive stimulus because increased levels of 5-HT and active signalling havebeen described in patients with PH [33]. More recently, 5-HT receptor overexpression was reported inpatients with IPF when compared with that in controls, particularly 5-HT receptor 2A in the pulmonaryartery smooth muscle layer [34]. In this study, we confirmed increased 5-HT receptor 2A expression inPAs from patients with IPF and 5-HT receptor 2A was significantly upregulated in patients with PH+IPF.5-HT receptor 2A initiates downstream signalling, including activation of RhoA/RhoA kinase (ROCK)and subsequent pulmonary vascular contraction. In this regard, the GC/cGMP/PKG pathway in smoothmuscle cells can decrease pulmonary vascular contractility by decreasing Ca2+ sensitivity andphosphorylating and inactivating the RhoA protein [35]. Thus, overexpression of PDE5 by decreasingcGMP and PKG may increase the activity of the RhoA/ROCK pathway, which increases pulmonaryvascular sensitivity to 5-HT, as suggested previously in pulmonary artery smooth muscle cells [36]. Thus,PDE5 overexpression in pulmonary arteries from patients with IPF and PH+IPF may partially explain whysildenafil selectively desensitised the contractile effects of 5-HT in these pulmonary arteries.

To understand further the effects of sildenafil in patients with PH+IPF, we analysed the molecular profileof pulmonary arteries. αSMA and vimentin gene expression was upregulated in pulmonary arteries frompatients with IPF and PH+IPF compared with those from donor subjects, and higher expression wasdetected in pulmonary arteries from patients with PH+IPF according to the PH remodelling profile.Furthermore, Col I and the profibrotic growth factor TGF-β1 were overexpressed in pulmonary arteriesfrom patients with IPF and PH+IPF and were more highly expressed in pulmonary arteries from patientswith PH+IPF, reflecting gradual activation of muscularisation and fibrosis. These results suggest that thevascular remodelling pathways are activated before PH is clinically detected, indicating the progressivenature of vascular remodelling in patients with IPF.

A similar molecular profile was recently described in pulmonary arteries from patients with PH+IPF usinggenome-wide expression profiling [37]. This profiling revealed a reduction in lumen diameteraccompanied by thickening of the media and intimal layers via an increase in extracellular matrixcomponents and αSMA in the intima and downregulation of von Willebrand factor exclusively localised inthe endothelium of pulmonary vessels [37]. In the present study, we also observed downregulation oftypical endothelial markers in pulmonary arteries from patients with IPF and PH+IPF, such asVE-cadherin, VEGFR-1, and FVIII, whereas eNOS was only downregulated in pulmonary arteries frompatients with PH+IPF, which may explain the endothelial-dependent relaxant effects of sildenafil in thepulmonary arteries from patients with IPF (figure 1b). We recently described loss of the endothelialphenotype and acquisition of the mesenchymal phenotype in endothelial cells from the pulmonary arteriesof patients with PH+IPF [16]. Recent in vivo data demonstrated that pulmonary capillary endothelial cellsmay be a source of fibroblasts in patients with pulmonary fibrosis via endothelial-to-mesenchymaltransition [4]. Therefore, a sildenafil-induced increase in the bioavailability of NO could improvepulmonary artery endothelial cell transformation and pulmonary remodelling. We previously observed thatadding sepiapterin in vitro increased levels of tetrahydrobiopterin (BH4) coupling of eNOS to produceNO, and thus inhibiting the endothelial-to-mesenchymal transition induced by TGF-β1 [16]. Otherstudies have demonstrated that chronic inhibition of eNOS induced endothelial-to-mesenchymal transitionin kidney endothelial cells [38].

In the present study, TGF-β1 increased the expression of mesenchymal markers and PDE5, and decreasedthe expression of endothelial markers in pulmonary artery rings from healthy donor subjects. A morerobust molecular change was observed in patients with IPF, indicating sensitisation to vascularremodelling. In both cases, sildenafil attenuated the increase in mesenchymal marker expression and thedecrease in endothelial marker expression. We isolated HPASMCs and HPAECs from the pulmonaryarteries of patients with IPF to dissect the possible mechanisms. TGF-β1 decreased endothelial andincreased mesenchymal marker expression in HPAECs, whereas TGF-β1 increased Col I and vimentin inHPASMCs, representing the myofibroblast transition. Sildenafil partially inhibited both the endothelial-and smooth muscle-to-mesenchymal transition by inhibiting ERK1/2 and SMAD3 phosphorylation, whichare the main downstream signals of TGF-β1. Although apparently unrelated, previous studies have shownthat NO inhibits TGF-β1/SMAD-regulated gene transactivation in a cGMP/PKG-dependent manner bydirecting proteasomal degradation of activated SMAD3 [39] or by sequestering SMAD3 [40]. ERK1/2phosphorylation can also be inhibited by PKG, which explains the inhibitory effects of sildenafil observedhere [36]. These findings are particularly relevant, as we observed hyperphosphorylation of ERK1/2 andSMAD3 in pulmonary arteries from patients with PH+IPF.

DOI: 10.1183/13993003.01259-2015 1747

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

We have provided the first evidence of direct relaxant/anti-contractile and anti-remodelling effects ofsildenafil on pulmonary arteries from healthy donors and patients with IPF or PH+IPF. Our findings maycontribute to understand the beneficial effects of sildenafil in patients with IPF; particularly in those withsevere PH. Future randomised controlled trials of sildenafil in this patient phenotype are warranted.

AcknowledgementsWe thank the personnel of the Dept of Pathology at the General University Hospital of Valencia and the animalhousing facilities of the University of Valencia, Valencia, Spain.

References1 Lettieri CJ, Nathan SD, Barnett SD, et al. Prevalence and outcomes of pulmonary arterial hypertension in

advanced idiopathic pulmonary fibrosis. Chest 2006; 129: 746–752.2 Heath D, Gillund TD, Kay JM, et al. Pulmonary vascular disease in honeycomb lung. J Pathol Bacteriol 1968; 95:

423–430.3 Blanco I, Ribas J, Xaubet A, et al. Effects of inhaled nitric oxide at rest and during exercise in idiopathic

pulmonary fibrosis. J Appl Physiol 2011; 110: 638–645.4 Hashimoto N, Phan SH, Imaizumi K, et al. Endothelial-mesenchymal transition in bleomycin-induced pulmonary

fibrosis. Am J Respir Cell Mol Biol 2010; 43: 161–172.5 Yeager ME, Frid MG, Stenmark KR, et al. Progenitor cells in pulmonary vascular remodeling. Pulm Circ 2011; 1:

3–16.6 Smith JS, Gorbett D, Mueller J, et al. Pulmonary hypertension and idiopathic pulmonary fibrosis: a dastardly duo.

Am J Med Sci 2013; 346: 221–225.7 Ghofrani HA, Wiedemann R, Rose F, et al. Sildenafil for treatment of lung fibrosis and pulmonary hypertension:

a randomised controlled trial. Lancet 2002; 360: 895–900.8 Zisman DA, Schwarz M, Anstrom KJ, et al. A controlled trial of sildenafil in advanced idiopathic pulmonary

fibrosis. N Engl J Med 2010; 363: 620–628.9 Han MK, Bach DS, Hagan PG, et al. Sildenafil preserves exercise capacity in patients with idiopathic pulmonary

fibrosis and right-sided ventricular dysfunction. Chest 2013; 143: 1699–1708.10 Hemnes AR, Zaiman A, Champion HC. PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and

pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation. Am J PhysiolLung Cell Mol Physiol 2008; 294: L24–L33.

11 Yildirim A, Ersoy Y, Ercan F, et al. Phosphodiesterase-5 inhibition by sildenafil citrate in a rat model ofbleomycin-induced lung fibrosis. Pulm Pharmacol Ther 2010; 23: 215–221.

12 Milara J, Juan G, Ortiz JL, et al. Cigarette smoke-induced pulmonary endothelial dysfunction is partiallysuppressed by sildenafil. Eur J Pharm Sci 2010; 39: 363–372.

13 Wharton J, Strange JW, Moller GM, et al. Antiproliferative effects of phosphodiesterase type 5 inhibition inhuman pulmonary artery cells. Am J Respir Crit Care Med 2005; 172: 105–113.

14 Ortiz JL, Milara J, Juan G, et al. Direct effect of cigarette smoke on human pulmonary artery tension. PulmPharmacol Ther 2010; 23: 222–228.

15 Milara J, Mata M, Mauricio MD, et al. Sphingosine-1-phosphate increases human alveolar epithelial IL-8secretion, proliferation and neutrophil chemotaxis. Eur J Pharmacol 2009; 609: 132–139.

16 Almudever P, Milara J, De Diego A, et al. Role of tetrahydrobiopterin in pulmonary vascular remodellingassociated with pulmonary fibrosis. Thorax 2013; 68: 938–948.

17 Jobse BN, Rhem RG, Wang IQ, et al. Detection of lung dysfunction using ventilation and perfusion SPECT in amouse model of chronic cigarette smoke exposure. J Nucl Med 2013; 54: 616–623.

18 Galie N, Humbert M, Vachiery JL, et al. 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonaryhypertension. Eur Respir J 2015; 46: 903–975.

19 Seeger W, Adir Y, Barbera JA, et al. Pulmonary hypertension in chronic lung diseases. J Am Coll Cardiol 2013; 62:25 Suppl, D109–D116.

20 Collard HR, Anstrom KJ, Schwarz MI, et al. Sildenafil improves walk distance in idiopathic pulmonary fibrosis.Chest 2007; 131: 897–899.

21 Zimmermann GS, von Wulffen W, Huppmann P, et al. Haemodynamic changes in pulmonary hypertension inpatients with interstitial lung disease treated with PDE-5 inhibitors. Respirology 2014; 19: 700–706.

22 Jackson RM, Glassberg MK, Ramos CF, et al. Sildenafil therapy and exercise tolerance in idiopathic pulmonaryfibrosis. Lung 2010; 188: 115–123.

23 Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 2004; 351:1425–1436.

24 Medina P, Segarra G, Martinez-Leon JB, et al. Relaxation induced by cGMP phosphodiesterase inhibitors sildenafiland zaprinast in human vessels. Ann Thorac Surg 2000; 70: 1327–1331.

25 Moreno L, Losada B, Cogolludo A, et al. Postnatal maturation of phosphodiesterase 5 (PDE5) in piglet pulmonaryarteries: activity, expression, effects of PDE5 inhibitors, and role of the nitric oxide/cyclic GMP pathway. PediatrRes 2004; 56: 563–570.

26 Pauvert O, Bonnet S, Rousseau E, et al. Sildenafil alters calcium signaling and vascular tone in pulmonary arteriesfrom chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 2004; 287: L577–L583.

27 Zhao L, Mason NA, Morrell NW, et al. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation2001; 104: 424–428.

28 Saleh D, Barnes PJ, Giaid A. Increased production of the potent oxidant peroxynitrite in the lungs of patients withidiopathic pulmonary fibrosis. Am J Respir Crit Care Med 1997; 155: 1763–1769.

29 Wiedemann R, Ghofrani HA, Weissmann N, et al. Atrial natriuretic peptide in severe primary and nonprimarypulmonary hypertension: response to iloprost inhalation. J Am Coll Cardiol 2001; 38: 1130–1136.

30 Song JW, Song JK, Kim DS. Echocardiography and brain natriuretic peptide as prognostic indicators in idiopathicpulmonary fibrosis. Respir Med 2009; 103: 180–186.

1748 DOI: 10.1183/13993003.01259-2015

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.

31 Baliga RS, Scotton CJ, Trinder SL, et al. Intrinsic defence capacity and therapeutic potential of natriuretic peptidesin pulmonary hypertension associated with lung fibrosis. Br J Pharmacol 2014; 171: 3463–3475.

32 Ried M, Potzger T, Neu R, et al. Combination of sildenafil and bosentan for pulmonary hypertension in a humanex vivo model. Cardiovasc Drugs Ther 2014; 28: 45–51.

33 Morrell NW, Adnot S, Archer SL, et al. Cellular and molecular basis of pulmonary arterial hypertension. J AmColl Cardiol 2009; 54: Suppl., S20–S31.

34 Konigshoff M, Dumitrascu R, Udalov S, et al. Increased expression of 5-hydroxytryptamine2A/B receptors inidiopathic pulmonary fibrosis: a rationale for therapeutic intervention. Thorax 2010; 65: 949–955.

35 Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol 2006; 68:345–374.

36 Li M, Sun X, Li Z, et al. Inhibition of cGMP phosphodiesterase 5 suppresses serotonin signalling in pulmonaryartery smooth muscles cells. Pharmacol Res 2009; 59: 312–318.

37 Hoffmann J, Wilhelm J, Marsh LM, et al. Distinct differences in gene expression patterns in pulmonary arteries ofpatients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis with pulmonaryhypertension. Am J Respir Crit Care Med 2014; 190: 98–111.

38 O’Riordan E, Mendelev N, Patschan S, et al. Chronic NOS inhibition actuates endothelial-mesenchymaltransformation. Am J Physiol Heart Circ Physiol 2007; 292: H285–H294.

39 Saura M, Zaragoza C, Herranz B, et al. Nitric oxide regulates transforming growth factor-beta signaling inendothelial cells. Circ Res 2005; 97: 1115–1123.

40 Gong K, Xing D, Li P, et al. cGMP inhibits TGF-beta signaling by sequestering Smad3 with cytosolicbeta2-tubulin in pulmonary artery smooth muscle cells. Mol Endocrinol 2011; 25: 1794–1803.

DOI: 10.1183/13993003.01259-2015 1749

PULMONARY VASCULAR DISEASES | J. MILARA ET AL.